“It is the weight, not numbers of experiments that is to be regarded.” Isaac Newton
Read any flow cytometry protocol and somewhere near the beginning will state something to the effect of ‘Place 1 million cells into a tube.’ The question is, faced with that special sample for THE experiment, how do you count cells to determine the number of cells in the tube? First we’ll give a bit of information on why it is important to know the number of cells you have.
Why Is It Critical to get Counting Cells to Know How Many Are in Your Tube?
1. Ensuring You Have Enough Cells to Perform the Experiment
Nothing is more disheartening than spending all day doing cell prep, staining and sorting only to realize that there are not enough cells to continue with your carefully planned experiment.
A back-of-the-envelope calculation can be very useful for ensuring you’ve got enough cells for your experiment. This calculation is:
Final Cells Needed x (1/sort efficiency) x (1/frequency) = Initial Starting Population
Table 1 shows this calculation for various frequencies.
Table 1. Example calculations for number of cells required.
|Final Cells Needed||Frequency of Population||Sorter Efficiency*||Input Cells|
|*Many sorters are more efficient than 50%, but it never hurts to err on the side of caution. If the cells of interest are fragile, reduce this efficiency.|
2. Cell Numbers Can Affect Staining
While the exact cell numbers may be less critical for some assays (surface staining, for example), the staining of cells using fluorescent probes (DNA, Redox, proliferation, etc.) are much more dependent on cell number for success.
3. Important for Measuring Magnitude of Effects
Counting cells can be very important in measuring the magnitude of the effect you are monitoring by flow cytometry. If an accurate count is not available, then these calculations are meaningless.
4. Cells Are Lost Through the Staining Process
Knowing the input cells at the beginning of the experiment and the output at the end of the experiment can determine cell loss, and allow for optimization of protocols to reduce cell loss. In intracellular staining, this loss can be as much as 70% of the input cells – so if 1 million cells are needed as input to the flow cytometer, one better start with staining 3 to 4 million cells.
What Are the Available Options for Counting Cells?
The tried and true method for counting cells is to use a hemocytometer. Invented by Louis-Charles Malassez, it is a specially etched microscope slide that holds a well-defined volume of sample. How to correctly use a hemocytometer was discussed in a previous Bitesize Bio post. If you are interested in even more information, check out the Hemocytometer.org blog.
The hemocytometer requires only minimal special tools (only the slide and a microscope), anyone can perform the assay with sufficient training, it can be used for counting cells a wide variety and types and makes use of the researcher as the visualization tool. Thus it remains the gold standard for counting cells.
Unfortunately, the hemocytometer has its limitations. The first is ensuring that the rules for counting cells are posted and agreed upon – the pattern of counting is critical to ensure that everyone gets the same answer. Second, it can require a great deal of training to ensure that the counting cells is done consistently (try getting a few of you lab mates to have a go at counting cells using the same hemocytomer and you might be surprised with the results!). A third limitation of the hemocytometer is the reliance on trypan blue for dead-cell discrimination. Finally as a manual method, it is very tedious for counting cells in anything more than just a few samples.
The other options are automatic systems for counting cells, which can be divided into three major categories: image-based technologies, impedance counting technologies (Coulter principle) and flow cytometry based methods.
Several vendors make instruments that do automated image cell counting. In general, these instruments use a microscope to capture an image of the field of view. Using the instrument-specific software, the system identifies cells based on diameter and does the cell counting based on that. They often also use Trypan Blue to exclude dead cells.
These systems, once optimized, are excellent for high throughput cell counting applications. One problem with these systems is that they usually require a specialized slide or cassette, which is an ongoing cost. A second issue is making sure that the system will recognize the size of cells of interest.
Impedance Counting Technologies
The Coulter Principle, first patented in 1953, is the basis of modern Complete Blood Counts (CBC) instruments, as well as the basis for the first cell sorter developed by Mack Fulwyler.
In principle, as a cell passes through an orifice separating two chambers filled with an electrolyte solution, there is a change in electrical resistance. This resistance change is proportional to the size of a cell. Some limitations of this technology include concentration of cells – too concentrated and the orifice will get clogged. Dead cells are identified based on size and changes in the impedance signal. Another issue is ensuring the electrolyte solution is correct. If the sample is not diluted in the proper solution, the impedance measurements will be off.
Beckman-Coulter and EMD Millipore have impedance counting technologies available. It is worth noting that the Millipore tool, the Scepter, is a hand-held instrument that looks like a pipet, making it easy to rapidly count multiple samples.
Flow Cytometry Based Technologies
Counting cells by flow cytometry can be performed in two different ways, depending on how the flow cytometer generates flow.
1. Pump/Syringe Systems
In the case of a pump/syringe type system (for example, the Accuri, Guava or MacsQuant), since the delivered volume is very precisely measured, an accurate event count can be made. With these systems, viability dyes and surface staining can ensure dead cells are excluded and specific subsets can be counted.
2. Differential Pressure Systems
For those cytometers that use differential pressure systems (e.g LSR-II or Gallios), since delivered volume is not accurately measured, a proportional counting bead can be used to enumerate cells. These beads are available from BD Bioscience, Life Technologies, and Spherotech among others. To use these beads, a sample is spiked with a defined volume of the beads. They are measured separately from the cells of interest, and based on the number of beads counted in the sample; a calculation can be made so that the number of cells in the sample can be determined. Of course, these beads will add a cost to the experiment.
Cell counting is critical for good flow cytometry. There are multiple ways to determine the number of cells in the sample, and each one has its strengths and weakness. From the gold standard of the hemocytometer to the automated technologies, there are ample ways to determine how many cells you have in the sample.
What’s your preferred method of cell counting?